In our studies, we have continued to examine the CTL response to peptide antigens presented by class I molecules of the Major Histocompatibility Complex (MHC), on the surface of tumor cells. Evidence has accumulated indicating that HLA-A2-restricted CTLs specific for human wild-type sequence p53 epitopes lyse tumor cells expressing mutant p53. Recently, in collaboration with Dr. DeLeo, Pittsburgh Cancer Center, we have used autologous dendritic cells(DC) pulsed with p53(264-272) peptide to generate p53 peptide-specific T cells from peripheral blood of patients with squamous cell carcinoma of the head and neck (SCCHN), as well as healthy donors. The generated T cells were specific for the p53(264-272) epitope, as determined by tetramer staining, and they efficiently recognized and killed antigen-expressing tumor cells. However, it was only possible to expand the CTL precursors from PBMC of SCCHN patients whose tumors are likely not to be recognized by these CTL. This finding suggests that in vivo CTL specific for p53 may well have influenced the outgrowth of "epitope-loss" tumors able to avoid these effector cells. The possibility that immunoselection of such tumor cells might occur during p53-based immunotherapy merits consideration in designing future clinical trials. It implies that the use of multiple epitopes might be necessary to prevent tumor escape from the immune system. To explore the possibility that wild-type sequence p53 peptides could also be used in vaccines for patients expressing HLA-A24 antigen, another frequent HLA class I allele, we investigated the induction of HLA-A24-restricted p53-specific CTLs from the peripheral blood lymphocytes of normal donors. Of six p53-derived peptides possessing an HLA-A24 binding motif, the p53(125-134) peptide was found to have a high binding capacity and induced peptide-specific CTLs from PBMC. A cloned CTL isolated from the bulk population by limiting dilution, lysed HLA-A24+ SCCHN targets more efficiently than the bulk CTL population. This finding indicates that vaccine strategies targeting wild-type sequence p53 epitopes can be extended to a wider range of cancer patients. In previous studies, we have demonstrated that the wild-type p53(264-272) peptide in complex with HLA-A2 is recognized by CD8+ T cells. In order to develop more effective vaccine strategies, a CD4+ T-cell-enriched population of PBMC obtained from a HLA-DR0401+ healthy donor was stimulated in vitro several times with autologous DC pulsed with human wild type p52 recombinant protein. We obtained a bulk population of CD4+ T cells which proliferated in response to T2-DR4+ target cells pulsed with the wild-type sequence p53(110-124) peptide. A CD4+ T-cell clone was isolated by limiting dilution from the bulk population and shown to be specific for the p53(110-124) peptide. These cells also proliferated in response to the PCI-13 SCCHN cell line as well as an HLA-DR4+ human melanoma cell line. These results indicate that wild type sequence p53(110-124) is a naturally presented HLA-DR4-restricted epitope of p53. Identification of this epitope will permit further studies aimed at detecting the presence of CD4+ T cells recognizing this epitope in PBMC obtained from cancer patients and the use of this CD4-defined epitope in cancer vaccine development. Presently, we are continuing work on the optimization of peptide epitopes for targeted immunotherapy, emphasizing the development of superagonist ligands in different systems other than MART1. We also are continuing our studies on crystal structures of T cell receptor (TcR)/MHC class I/peptide complexes having partial agonist, antagonist, and superagonist activity.